Cryoprotectant role of exopolysaccharide ID1 in the vitrification/in-straw warming of in vitro-produced bovine embryos.

The cold-adapted bacterium Pseudomonas sp. ID1 produces the extracellular exopolysaccharide ID1 (EPS ID1) with cryoprotective activity. This study was designed to optimize the vitrification/in-straw warming protocol of in vitro-produced (IVP) blastocysts by adding EPS ID1 to the vitrification media. Day 7-expanded blastocysts were vitrified/warmed using the VitTrans device after the addition of 0 or 100 μg/mL EPS ID1 to the vitrification media. Blastocysts vitrified by the Cryotop method and fresh non-vitrified blastocysts served as controls. Outcomes were assessed in the warmed embryos in terms of survival rates and mRNA relative abundances of BAX, BCL2, GPX1, and CDX2 genes. No differences in survival rates were observed at 3 h post-warming between vitrification treatments. At 24 h post-warming, the addition of EPS prior to vitrification with the VitTrans device produced similar survival rates to Cryotop-vitrified embryos and similar hatching rates to fresh non-vitrified or Cryotop-vitrified embryos. No differences emerged in BCL2 gene expression. Lower BAX (p < .05) and higher GPX1 (p < .05) and CDX2 (p < .1) gene expression were observed in expanded and/or hatched blastocysts derived from VitTrans-EPS-vitrified embryos when compared to those from the non-supplemented group. In conclusion, addition of EPS not only promoted blastocyst survival and hatching after VitTrans vitrification/warming but also modified the expression of genes associated with better embryo quality.

Martínez-Rodero I ，Salas-Huetos A ，Ordóñez-León A ，Hidalgo CO ，Yeste M ，Mercadé E ，Mogas T ... -  《-》

Exopolysaccharide ID1 Improves Post-Warming Outcomes after Vitrification of In Vitro-Produced Bovine Embryos.

Ordóñez-León EA ，Martínez-Rodero I ，García-Martínez T ，López-Béjar M ，Yeste M ，Mercade E ，Mogas T ... -  《INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES》

Vitrification and in-straw warming do not affect pregnancy rates of biopsied bovine embryos.

In the cattle industry, in vivo or in vitro embryo production combined with genotyping and cryopreservation technologies allows the selection and conservation of embryos carrying genes for desirable traits. This study aimed to assess the efficiency of a vitrification method suitable for in-straw warming of biopsied in vivo derived (IVD) bovine embryos. Three experiments were carried out using two methodologies: the Cryotop®, the gold standard vitrification and 3-step warming methodology, or the VitTrans, a vitrification/in-straw 1-step warming method that enables direct embryo transfer to the uterus. In experiment 1, intact and biopsied in vitro produced (IVP) day 7 expanded blastocysts were vitrified using the Cryotop® and warmed in 1- or 3-steps. No differences in survival rates were recorded at 24 h after warming for intact or biopsied IVP blastocysts irrespective of the warming procedure. In experiment 2, the effect of the time from trophectoderm (TE) biopsy to vitrification/in-straw warming on post-warming survival rate was assessed. No significant differences in survival were observed when blastocysts were vitrified/in-straw warmed immediately after biopsy or after 3 h in culture when compared to intact blastocysts. In experiment 3, IVD embryos were vitrified 3 h after biopsy using the Cryotop® or the VitTrans method and pregnancy rates were assessed at day 60 after transfer. Fresh, biopsied embryos served as control. Similar pregnancy rates were observed when IVD biopsied embryos were transferred fresh or vitrified/warmed by the Cryotop® or VitTrans method. No significant effect of the embryo quality or developmental stage was detected on the percentage of pregnant recipients when IVD biopsied embryos were transferred fresh or after vitrification. While fresh female IVD embryos produced significantly higher pregnancy rates than male embryos, there were no differences in pregnancy rates when male or female vitrified/warmed embryos were transferred. About 81% from the biopsies analyzed successfully determined the embryo sex, confirming that DNA was there, and it was efficiently amplified. To conclude, our findings indicate that both vitrification methodologies produced similar post-warming outcomes for both intact and biopsied IVP embryos. Besides, vitrification/in-straw warming of biopsied IVD bovine embryos did not affect the viability to originate pregnancy, being a useful option for their direct transfer in field conditions.

González-Rodríguez N ，Martínez-Rodero I ，Scherzer J ，Jung S ，Reichenbach M ，Zablotski Y ，Otzdorff C ，Zerbe H ，Mogas T ... -  《-》

Survival of vitrified in vitro-produced bovine embryos after a one-step warming in-straw cryoprotectant dilution procedure.

Vitrification is an alternative to slow-rate freezing for cryopreserving bovine embryos. However, this technology requires simplification if it is to be used under field conditions. The main objective of this work was to develop a new system for the direct transfer of vitrified embryos to be used under farm conditions. For this, three objectives were set: (1) to compare the effect of vitrification, using the cryologic vitrification method (CVM), and slow-rate freezing on bovine embryo development and quality; (2) to develop a one-step warming procedure for bovine in vitro-produced (IVP) vitrified (by CVM) embryos; and (3) to assess the effects on embryo survival of a new method for the direct transfer of vitrified IVP bovine blastocysts. In vitro-produced blastocysts were initially either vitrified by CVM or subjected to slow freezing to compare embryo survival and quality (experiment 1). No differences were detected between these cryopreservation techniques in terms of the survival and quality variables at 24 hours or in terms of the proteins expressed. However, at 48 hours the vitrified embryos showed higher hatching rates, greater total cell numbers, and lower apoptotic indices (P < 0.05). In experiment 2, CVM-vitrified IVP blastocysts were warmed by the conventional two-step or one-step warming procedure by incubating them at 41 °C in 0.25 M sucrose for 10 minutes, 0.15 M sucrose for 10 minutes, or 0.25 M sucrose for 5 minutes. In addition, embryo transfer (ET) was performed using vitrified embryos warmed by the one-step procedure in 0.25 M sucrose solution for 5 minutes. As a control group, IVP fresh embryos were transferred to recipient females. No differences were observed in embryo survival or total cell number between any of the warming procedures. Moreover, no significant differences for pregnancy at 60 days were found between the ET groups. In experiment 3, expanded IVP blastocysts were then either vitrified using a conventional or a modified fiber plug designed to allow direct ET after in-straw cryoprotectant (CP) dilution. They were warmed using the one-step process (0.25 M sucrose, 5 minutes) in a 0.25 mL French straw. Embryo recovery associated with the modified fibreplug system was less reliable than with the conventional system. However, no differences were seen between the systems in terms of in vitro embryo survival among those finally recovered. Finally, IVP blastocysts were vitrified using conventional fibreplugs to maintain a high embryo recovery rate, and then warmed using the one-step warming in-straw CP dilution procedure, but using an adapter with a wider opening coupled to the French straw and a heated metal chamber to protect and keep the straw at 41 °C (experiment 4). No differences were seen in embryo survival rates between the two groups. The CVM combined with this new one-step warming in-straw CP dilution procedure could be used for direct ET under field conditions.

Caamaño JN ，Gómez E ，Trigal B ，Muñoz M ，Carrocera S ，Martín D ，Díez C ... -  《-》

Cryoprotectant role of exopolysaccharide of Pseudomonas sp. ID1 in the vitrification of IVM cow oocytes.

Arcarons N ，Vendrell-Flotats M ，Yeste M ，Mercade E ，López-Béjar M ，Mogas T ... -  《-》